1. Analysis of Lunch Meat Microbial Contamination
Experiment and Analysis by Donald Kline, Jr

2. Problem of providing safe food
Throughout history, people have been faced with the challenge of not just providing food, but keeping it safe from contamination

3. Problems of having safe meat
Meat is especially susceptible to disease because it came from a living animal with natural flora like E. coli in addition to diseases the animal could have contracted during its life
The nutrients in meats provide can provide an excellent growth
source for microbial life

4. Pathogens
Pathogens in food are the leading cause of death in some undeveloped countries, with a death toll of around two million people annually
Medical care for pathogens costs over a billion dollars per year worldwide

5. Common contaminants in meat
E. Coli
Salmonella
Clostridium botulinum
Campylobacter jejuni
Listeria monocytogenes
Clostridium perfringens
Staphylococcus aureus

6. Historical ways people kept meat safe
Until 1860, it was thought that food became spoiled through spontaneous generation
Drying using the sun is the oldest form of food preservation
Salting can be an effective means of preserving meat
Smoking and heating were other common ways to keep food safe
Meats usually could not be kept safe from contamination very long

7. Common ways to keep meat safe
Keep the meat in a hypertonic environment
Sterile packaging
Cooking
Refrigeration
Salting
Freeze-Drying
Canning and Bottling

8. Preservatives
A preservative is used to prevent spoilage in foods from microbial growth and contamination
Common chemical preservatives are calcium propionate, sodium nitrate, sodium nitrite, sulphites, disodium EDTA, formaldehyde, glutaraldehyde, and methylchloroisothiazolinone
Natural preservatives include salt, sugar, vinegar, and diatomaceous earth. Citric and ascorbic acids can also assist in the preservation of certain foods.

9. Preservatives in this experiment
Virginia Brand ham contained water, sugar, salt, dextrose, potassium chloride, sodium phosphate, sodium erythorbate, sodium nitrite, and sugar
Giant Eagle contained the following preservatives: water, salt, sugar, dextrose, sodium phosphates, sodium erythorbate, and sodium nitrite.
Russer ham contained water, salt, dextrose, sodium nitrate, sodium phosphate, and sodium erythorbate.

10. E. Coli
E.Coli is a pathogen that is found in the lower intestines of warm blooded animals
Represents prokaryotic cell model in this experiment
There are almost 73,000 cases of infection and 61 deaths per year in the United States

11. Yeast
Type of yeast used in this experiment was Saccharomyces cerevisiae
Yeasts also contribute to food spoiling by producing waste products when they metabolize food
Yeasts contribute to making many foods we use today
They represent the eukaryotic cell model in this experiment

12. Purpose and Hypothesis
Determine how different brands of sterilized lunchmeat’s preservatives and ingredients would affect the survivorship of yeast and E. coli cells
The null hypothesis was that the lunch meats would not significantly affect survivorship of S. cerevisiae and E. coli cells

13. Materials
96 LB agar plates( 1.5 % tryptone, .5 % yeast extract, 1% NaCl, 1.5 % agar)
LB media (1 % tryptone, 5 % yeast extract, 1% NaCl)
72 YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar)
Sterile dilution fluid (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, .1mM CaCl2, 100mM NaCl)
Klett spectrophotometer
Sterile pipette tips and Micropipettors
Vortex
Incubator
Sidearm flask
Spreader bar
Ethanol
20 mL Sterile capped test tubes
E.coli B
Saccharomyces cerevisiae
Giant Eagle Old Fashioned Ham, Russer Cooked Ham,
and Dietz and Watson Virginia Brand Ham
Hole puncher
Metric Scale and weigh boat
Micro burner

14. Procedure
E. coli B and Saccharomyces cerevisiae were grown over night in a sterilized media
A sample of the overnight cultures were added to separate fresh LB (bacteria) and YEPD (yeast) in a sterile sidearm flask.
The cultures were incubated at 37°C (bacteria) and 30°C (yeast) until a density of 50 Klett spectrophotometer units was reached. These represent cell densities of approximately 108 (bacteria) and 107 (yeast) cells per mL.
The cultures were diluted in sterile dilution fluid to a concentration of approximately 105 cells per mL.
The hams were sliced, massed to .4 grams for each brand of ham and sterilized separately by soaking in 95% ethanol. After the ethanol was evaporated, the hams were placed in 15mL sterile polystyrene conical tubes.
100uL of the 105 cells/mL cell suspensions were pipetted directly onto the surface of the ham pieces.

15. Procedure 2
The tubes were allowed to incubate at room temperature for the following time periods: 0, 45, 90, and 135 minutes (bacteria) and 0, 30, and 60 minutes (yeast).
One mL of SDF was pipetted onto the ham/cell mixture and the tubes were gently vortexed.
After vortexing to evenly suspend cells, 100uL of the cell suspension was transferred to 9.9mL of SDF and then the SDF was vortexed and 100 uL aliquots were then spread onto either LB agar or YEPD agar.
The plates were incubated for 24 hours at 37°C (bacteria), or 48 hours at 30°C (yeast).
The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

16. 100 uL
100 uL
100 uL
108 cells/mL (bacteria) or 107 cells/mL (yeast)
1mL of SDF
105 cells/mL for both yeast and bacteria
105 cells/mL with ham
103 cells/mL
102 cells

18. p Values for Bacteria Control Virginia G. Eagle Russer 0 min Bac
Control
Virginia
G. Eagle
Russer
0 min Bac
p › .05
45min Bac
1.35E-10
p ‹ .01
p ‹ .01
90min Bac
1.1E-12
135min Bac
6.83E-13

19. Conclusions for Bacteria
Only Giant Eagle Ham had a significant negative impact on E. coli survivorship.
Russer Cooked Ham appeared to have the least negative impact on E. coli survivorship. The variation was not statistically significant.
Giant Eagle Old Fashioned Ham appeared to have the most negative impact on bacterial survivorship

21. Data Chart of p values for yeast
Control
Virginia
G. Eagle
Russer
0 min Yeast
0.0258
p › .05
p ›.05
30min Yeast
60min Yeast
p ‹ .05

22. Yeast Conclusions
Giant Eagle ham had a statistically significant negative impact on yeast survivorship, while Russer cooked ham and Virginia Brand Ham did not
Russer Cooked Ham appeared to have the least negative impact on yeast survivorship. The variation was not statistically significant.

23. Limitations and Extensions
In the process of sterilizing the hams, it might have affected the composition of the preservatives
The moisture content of the hams might have varied due to the sterilizing and drying processes
Some hams might retain the bacteria or yeast on the surface longer than others
Infuse the meats directly into agar to allow longer exposure times for the cells
Sterilize ham with gamma irradiation
Use different models of bacteria and eukaryotic cell models

24. Sources Centers for Disease Control and Prevention (CDC)
Food and Drug Administration (FDA)
Partnership for Food Safety Education (PFSE)
World Health Organization (WHO)
Acknowledgement to Professor John Wilson University of Pittsburgh Biostatistician for his advice and statistical analysis